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Assessment of using dried vinasse rice to replace soybean meal in lambs diets: In vitro, lambs performance and economic evaluation
Small Ruminant Research 173 (2019) 1–8
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Assessment of using dried vinasse rice to replace soybean meal in lambs diets: In vitro, lambs performance and economic evaluation
T
⁎
Hani M. El-Zaiata, , Diogo D. Réb, Harold O. Patinob, Sobhy M.A. Sallama a
Department of Animal Production, Faculty of Agriculture, University of Alexandria, Aflaton St., El-Shatby, P.O Box 21545, Alexandria, Egypt Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 7712 Bairro Agronomia CEP: 91540000, Porto-Alegre, RS, Brazil
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dried vinasse rice Rumen fermentation Performance Soybean meal Texel lambs
Effects of replacing soybean meal (SBM) with dried vinasse rice (DVR) as an alternative source of protein were evaluated under in vitro and in vivo conditions. Protein from SBM was partially replaced by DVR protein at the levels of zero (DVR0) control, 250 (DVR250), 500 (DVR500) and 750 g (DVR750) of the original concentration. An in vitro semi-automatic system was employed to evaluate gas production, feed degradability and fermentation profile of diets. The ANOVA and regression coefficients indicated that the DVR effective level was 500 g/kg DM. An in vivo experiment on twelve Texel lambs (BW; 30.48 ± 0.45 kg) kept individually in wooden pens and allotted into randomized complete block design with 6 blocks by 2 treatments as: control (diet without DVR) and DVR (500 g/kg DM) lasted for 60 days, and the subsequent 7 days were assigned for the digestibility trial. Upon increasing dietary DVR, net CH4 production decreased quadratically (P < 0.05). Ruminal NH3-N concentration and total protozoal count decreased linearly (P < 0.05) as DVR levels increased. In vitro microbial protein and propionate proportion (C3) increased quadratically (P = 0.027 and P = 0.034, respectively), while total short chain fatty acids, acetate (C2) butyrate, isobutyrate and C2:C3 ratio decreased quadratically (P = 0.036, P = 0.020, P = 0.029, P = 0.046 and P = 0.042, respectively). Lambs fed 500 g DVR/kg DM showed higher (P < 0.001) daily gain, body weight, feed efficiency and crude protein digestibility compared to control. The DVR diet showed higher (P < 0.05) digestible, metabolizable and net energy than control. DVR500 diet reduced (P < 0.001) feed costs and increased (P < 0.001) the net profit compared to control. The use of DVR provides a promising source of both protein and energy for growing lambs with extra potentials for economic advantages without adverse effects on growth performance.
1. Introduction
double productivity (14 ton/ha) have been cultivated and are not used for human consumption but for ethanol production and animal nutrition (EMBRAPA, 2013). Vinasse, a co-product obtained after distillation of fermented materials can be used as a source of nutrients to support ruminant performance (López-Campos et al., 2011; Rodrigues and Hu, 2017). This could allow for more value added to the ethanol production chain and is more environmentally sustainable (Barros et al., 2009). Addition of 13% vinasse to dried sugar beet pulp improves disappearance rates of dry matter with no effect on ruminal pH, ammonia concentration, or individual and total short chain fatty acids (Fernández et al., 2009). Further, inclusion of 5% vinasse in the diet showed positive effects on nitrogen supply and modified positively body composition of growing calves (Zali et al., 2017). Feeding 100 and 200 g of vinasse/kg of concentrate mixture increased ruminal pH and reduced feed intake while increased the feed to gain ratio in fattening lambs
Soybean meal (SBM) represents the most frequently used source of dietary protein in livestock feeding, however, it is one of the most expensive ingredients. Sheep owners in the developing countries are not always able to supplement their lambs concentrate mixtures with SBM, though it is necessary to meet the nutritional demands of lambs during the early growth phase. Therefore, looking for low price protein source as an alternative to SBM, while keeping reasonable nutritive value to reduce dietary cost and increase economic profits is a must. New technologies to produce ethanol from different plants raw materials such as sugarcane, corn and other feedstocks are available and applicable in Brazil (Lópes et al., 2016). Meanwhile, challenges facing biofuel production chain seek an efficient methodology to get rid of the generated co-products (Zali et al., 2017). New rice cultivars with
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Corresponding author. E-mail addresses: [email protected], [email protected] (H.M. El-Zaiat).
https://doi.org/10.1016/j.smallrumres.2019.01.003 Received 11 July 2018; Received in revised form 17 November 2018; Accepted 14 January 2019 Available online 14 February 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
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ground corn, SBM and minerals were formulated for in vitro assay evaluation. The control diet contained 36 g SBM/kg DM as the main source of protein and was free of DVR (DVR0). In the remaining three DVR diets, protein from SBM was partially replaced by DVR protein at the levels of 250 (DVR250), 500 (DVR500), and 750 g (DVR750) of the original SBM amount which corresponded to 0, 13, 26, and 39 g of DVR, respectively, of the dietary DM. The feed ingredients and chemical composition of the experimental diets are presented in Table 2. The readings of GP pressure in the bottles headspace were taken using semi-automatic system procedure equipped with a pressure transducer and a data logger (Pressure Press Data GN200, Sao Paulo, Brazil) according to Bueno et al. (2005). To avoid the effects of unusual ruminal conditions, six adult rumencannulated Barki sheep (BW, 49.0 ± 2.3 kg) were used as rumen content donors. Animals were fed Berseem (clover) hay ad libitum plus 0.750 kg/100 kg BW of commercial concentrate mixture (145 g CP/kg DM) plus mineral premix to meet nutrients requirement (NRC, 2007). Ruminal liquid and solid fractions (500:500 v/v) were collected separately from each animal before morning feeding and placed separately into thermo containers (preheated with water at 39ºC) under anaerobic conditions and transported immediately to the laboratory. The two fractions were blended at 1:1 (v/v) for 10 s, strained by hand through four layers of cheesecloth before combining with buffer solution, then held under CO2 to ensure anaerobic conditions in a water bath (39ºC) until used (Bueno et al., 2005). Each treatment was incubated in three inocula, each prepared from two animals. This was necessary due to the large amount of inoculum needed and to ensure that the natural variation among animals was maintained and at the same time, the effects of unusual rumen inocula or rumen environments were reduced. For each inoculum, four bottles per treatment were prepared: two for in vitro truly degraded organic matter (IVTDOM) and in vitro truly degraded dry matter (IVTDDM) determination and the other two bottles for determining fermentation constituents. A corresponding four bottles consisted only of rumen fluid and buffer solution without substrate (blank) to correct the GP from the inoculum, and further, four bottles contained Berseem hay as an internal standard to correct the variation between inoculum. The total bottles of the procedure were: 72 bottles ((4 replicates × 4 treatments+ 4 internal standard + 4 blanks) × 3 inoculum) for all inocula. A representative substrate sample was ovendried and ground in a Wiley mill to pass through a 1 mm screen for use in the incubations assay. A 300 mg of dried substrate were accurately weighed into 120 mL glass bottles and stored overnight at 39ºC. Each bottle (pre-warmed to 39ºC) was then injected with 30 mL of MB9 medium (Onodera and Henderson, 1980) and 15 ml of rumen inoculum. The pH was adjusted to 6.8 and CO2 was flushed for 30 min. After filling, bottles were then closed with a rubber stoppers (Bellco Glass Inc, Vineland, NJ, USA), manually shaken to mix and placed in a forced air incubator at 39ºC (FALC instruments S.R.L., Treviglio, Italy) for 24 h. The gas headspace pressure (75 mL) was recorded at distinct incubation times 6, 12 and 24 h from the beginning of incubation using a pressure transducer. The gas volume (mL) = 4.974 × measured pressure (psi) + 0.171 (n = 500; r2 = 0.99; data are not reported). For the CH4 analysis, representative gas samples of 2 mL were collected from the bottles at distinct incubation times using 5 mL syringe (med Dawliaico, Assiut, Egypt) and accumulated in 10 mL Vacutainer tubes (BD Vacutainer® Tubes, NJ, USA) to determine the CH4 concentration. After each gas sampling, the bottles were vented to release the gas pressure accumulation, shaken manually and immediately returned to the incubator oven thereafter. The CH4 concentration was determined by gas chromatography (Model 7890, Agilent Technologies, Inc, Colorado 80537, USA) with three valve system using 1/8 inch packed columns having early back flush of the C6 components and equipped with a thermal conductivity detector. Separation was achieved using micro packed column using helium as carrier gas with a flow rate of 28.0 mL/min. The detector and
(López-Campos et al., 2011). The limitation of exploiting vinasse in animal feeds is associated with its high-water contents which make it difficult to handle, transport and store. Historically, several studies have been conducted to evaluate different types of wet vinasse, but no definitive results were accomplished for the utilization of dry vinasse as a co-product obtained from rice fermentation in animal feeding. We hypothesized that inclusion of dried vinasse obtained from the alcoholic fermentation of rice (DVR) can improve the dietary nutritive value, digestibility and animal performance. Therefore, the objectives of this study were initiated to evaluate the effect of DVR as an alternative source of protein on in vitro ruminal fermentation, in vivo apparent digestibility, growth performance and feed costs of Texel sheep. 2. Materials and methods The in vitro evaluation assays were conducted at Laboratory of Animal Nutrition, Department of Animal Production, Faculty of Agriculture, Alexandria University, Egypt. The in vivo experiment was carried out at ruminant sector of Laboratório de Ensino Zootécnico, Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul (UFRGS), Brasil (30°_04_21_21 S 51°_08_13_88 N). All experimental procedures involving the use of animals were approved by ethical committee for care and use of agricultural animals in agricultural research and teaching experiments obtained from UFRGS, Brazil. 2.1. Preparations of dried vinasse rice (DVR) The vinasse co-product was obtained from the fermentation process of rice grains. After removing the hulls rice grains are ground to 400 μm, saccharified, fermented, distilled with alcohol plates in a distillation column system and the vinasse is separated, thereafter, dried in a forced-air oven at 40 °C for 72 h. The mineral composition and characteristics of DVR are presented in Table 1. 2.2. In vitro gas production (GP) evaluation assay (Experiment 1) In vitro batch culture assay (Experiment 1) was employed to evaluate the effects of replacing dietary soybean meal (SBM) with increasing levels of dried vinasse rice (DVR) incubated at 39 °C for 24 h and to select the effective level of DVR on nutrients digestibility as well as lambs performance (Experiment 2). The experimental diet used as a fermentation substrate was formulated (a mixture of forage and concentrate 75:25, w/w) as presented in Table 2. The basal experimental diet (127 g CP /kg of DM) contained 750 g/kg DM chopped Oats (Avena Strigosa) hay and 250 g/kg DM concentrate mixture consisting of Table 1 Minerals composition and characterization of experimental dried vinasse rice (DVR). Items characterization pH Organic carbon, g/kg Nitrogen, g/kg DM Gross energy, Mcal/kg DM Minerals, g/kg DM Phosphorus Potassium Calcium Sodium Magnesium Sulfur Trace elements Copper Zinc Manganese
Quantity
3.8 470 48 5.12 7.10 5.00 0.80 0.43 2.50 3.70 0.018 0.070 0.064
2
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Table 2 Feed ingredients and chemical composition of experimental diets on DM basis. Items
Ingredients Oats hay Maize grains Soybean meal (SBM) Dried vinasse rice (DVR) Limestone Mineral and vitamin mixturesa GE, Mcal/kg DM ME, Mcal/kg DMb NEm, Mcal/kg DMb NEg, Mcal/kg DMb Chemical composition, g/kg DM OM CP EE NFCc NDFom
Oats hay
SBM
DVR
Maize grain
Replacement levels (g DVR/kg SBM) 0
250
500
750
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ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
750 210 36 0 1.50 2.50 3.38 2.77 1.84 0.45
750 206 27 13 1.50 2.50 3.45 2.83 1.89 0.49
750 202 18 26 1.50 2.50 3.51 2.88 1.93 0.53
750 198 9 39 1.50 2.50 3.60 2.95 1.99 0.58
842 119 26 244 452
936 441 34 352 154
960 301 155 453 123
987 107 37 817 165
872 128 28 337 379
873 127 30 331 379
873 127 32 331 378
873 127 33 332 378
a Composition: Each one kg consisting of Mn, 75 mg; Zn, 70 mg; Se, 300; Fe, 50 mg; Cu, 8 mg; I, 750 mg; Vit A, 8000 IU; Vit D3, 2000 ICU; Vit K3, 1.8 IU; Vit B1, 1.8 mg; Vit B2, 6 mg; Vit B12, 12 mg; Vit B6, 2.8 mg; thiamine, 3 mg; folic acid, 1.0 mg; Biotin, 60 mg; Pantothenic acid, 10 mg; nicotinic acid and 40 mg. DVR: dried vinasse rice; GE: gross energy; ME: metabolizable energy; NEm: net energy for maintenance; NEg: net energy for gain; DM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; NDFom: neutral detergent fibre expressed exclusive of residual ash. b Calculated according to NRC (2001). c NFC: non-fibre carbohydrates (g/kg) = 1000 − (NDFom g/kg + CP g/kg + EE g/kg + ash g/kg).
described by Konitzer and Voigt, (1963). Aliquots of 2 ml subsample of ruminal fluid were mixed with 4 ml of methyl green formalin-saline solution, preserved in glass bottle and stored in darkness at room temperature until total protozoa enumeration (El-Zaiat et al., 2018). For protozoa counting, bottles were mixed well immediately before counting and about 10 μl of the sample were loaded onto a counting chamber (Neubauer Improved Bright-Line counting cell, 0.1 mm depth; Labor Optik, Lancing, UK) and held on an optical microscope with an objective 45x/.66 according to the procedure described by Dehority (1993). The short chain fatty acids (SCFA) concentration was determined using GC as described by Abo-Zeid et al. (2017) using a gas chromatograph (Thermo Fisher Scientific, Inc., TRACE1300, Rodano, Milan, Italy) fitted with an AS3800 autosampler and equipped with a capillary column FFAP (260 N 225 P; 0.5 μm film o.d., 0.53 μm i.d., and 30 m length; J and W Agilent Technologies Inc., Palo Alto, CA). Nitrogen at 7 mL min−1 was used as carrier gas. Air, hydrogen and nitrogen fluxes (make up gas) were kept at 450, 40, and 35 mL/min, respectively. A 0.1 μL aliquot was injected in splitless mode for the entire run with 31.35 mL min−1 of H2 flux (63.432 Pa). Injector and flame ionization detector (FID) temperatures were held isothermally at 250 °C. Oven heating slope was 80 °C (1 min), 120 °C (20 °C min−1 for 3 min), and 205 °C (10 °C min−1 for 2 min), with 9 min overall analytical time.
column temperatures were 250 °C and 60 °C, respectively. The test of linearity and calibration were accomplished using a standard gas curve in the range of probable concentrations of the samples. The produced CH4 was calculated according to the method of Longo et al. (2006) as follows: CH4, ml = (Total GP, ml + Headspace, 75 ml) × CH4 concentration (ml/ml). The net GP and CH4 values were determined by correcting for the corresponding blank values.
2.2.1. In vitro degradablity and fermentation constituents After 24 h of incubation, the fermentation process was inhibited by swirling all bottles in ice. For determining IVTDOM and IVTDDM, bottles were injected with 70 ml of neutral detergent solution immediately and incubated at 105ºC for 3 h, assuming complete removal of microbial cells by the detergent as described by Blümmel and Becker (1997). Bottles contents were filtered, washed with hot water (50 °C) and then acetone and transferred into pre-weighed crucibles, and residual DM was estimated. The residual DM left above was presented as IVTDDM and calculated as following: = {(g Sample DM – g Residue DM − g Blank DM)/g incubated substrate}*1000. The dried residual was then ashed at 550 °C to calculate IVTDOM as following: = {(g Sample OM – g Residue OM − g Blank OM)/g incubated substrate}*1000
2.3. Animals and experimental design (Experiment 2) Twelve castrated Texel crossbred lambs (age 12 months, initial body weight, IBW 30.48 ± 0.45 kg) individually housed in wooden pens were allotted into completely randomized block design with 6 blocks and 2 dietary treatments; control (basal diet without DVR) and DVR (500 g/kg DM). The experiment lasted for 60 days to evaluate lamb performance, whereas the subsequent 7 days were assigned for the nutrient digestibility trial. The pens were equipped with feeders and water buckets for drinking. Two weeks before the onset of the experiment, lambs were treated for internal parasites with oral administrations of levamisole chloridate (Ripercol; Fort Dodge Saúde Animal® LTDA, Campinas, SP, Brazil) as anthelmintic according to BW.
The microbial crude protein (MCP) was calculated according to Menke et al. (1979) as: MCP (mg/kg TDOM) = TDOM, g/kg incubated – (net GP, mL × 2.2) where 2.2 (mg/mL) is the stoichiometric factor which expresses organic matter (mg) required for SCFA gas combined with one mL of GP (Blümmel et al., 1997). The liquid phase of the other two bottles was used for the fermentation constituents and protozoal count assessments. Ruminal fluid pH was measured immediately after collection using a portable pH meter (GLP 21 model; CRISON, Barcelona, Spain). The ammonia (NH3-N) concentrations were determined colorimetrically by spectrophotometer (Alpha-1101 model; Labnics Equipment, California, USA) using commercial lab test (SPINREACT, Ctra. Santa Coloma, 7, Girona, Spain) as 3
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(2017) using the following formula = (market price of BW × ADG) − FCD where the market cost of BW is 1.5 US$/kg and FCD is the feed cost per day. The market feed price (MFP) of oat hay, maize grain, SBM and DVR were 0.10, 0.15, 0.33 and 0.09 US$/kg respectively. Economic efficiency was calculated as output/input: (ADG × 1.5)/(DMI × MFP) to elucidate the practical efficiency of DVR on animal performance.
2.3.1. Performance and feeding management The total mixed ration (TMR) diet consisted of 750 g/kg chopped oats (Avena Strigosa) hay and 250 g/kg concentrate (75:25 forage:concentrate, F:C). Lambs were fed TMR diet twice a day at 0900 and 1700 h in equal portions allowing extra refusal amount of approximately 10% of the daily DMI to meet their nutritional requirements (NRC, 2007). Lambs were allowed feed ad libitum with free access to fresh water throughout the experiment. The amounts of feed offered and refused were recorded daily to estimate feed dry matter intake (DMI) as the difference between daily feed supply and residues. Weekly feed samples were collected to adjust dietary F:C ratio and to be kept for subsequent analyses. Animals were weighed every 30 days before the morning feeding after 16 h fasting in order to monitor body weight changes. Body weight gain (BWG) was calculated as the difference between final body weight (FBW) and IBW; average daily gain (ADG) was the total BWG divided by 60-days; feed efficiency (FE) was obtained by dividing ADG by DMI.
2.6. Statistical analysis In the in vitro assay, each experimental treatment was incubated in duplicate (analytical replicates) and completed in one run for all treatments, using three inocula (statistical replicates) (6 values per treatment). Prior to statistical analysis, in vitro analytical replicates data from the different bottles were averaged. Data from in vitro experiment were analyzed using the PROC MIXED procedure of SAS (version 9.1, SAS Inst. Inc., 2002, Cary, NC). The statistical model included the fixed effect of diet DVR levels (i = DVR0, DVR250, DVR500 and DVR750). In vitro assay data were subjected to one-way ANOVA and regression to test the effects of incremental levels of DVR for SBM on the measured variables. However, because there was no cubic effect, only the DVR linear and quadratic effects are discussed. Growth performance data were analyzed using the MIXED procedure for a randomized complete block design according to the model: Yijk = μ + Ti + Pj + Bk + PTij + еijk where Yijk = observations mean, μ = overall mean, Ti = fixed effect of DVR levels (i = DVR0 and DVR500), Pj= fixed effect of period (j = 1 or 4), Bk = random effect of block (k = 1 to 6), PTij= interaction of DVR × period and eijk = residual error. The lamb nutrients digestibility and economic efficiency were analysed excluding the random effect of block: using the following statistical model as Yi = μ + Ti + ei, where Yi = observations mean, μ = overall mean, Tj = treatment effect (j = 1 to 2), and ei = residual error. Statistical significance level was declared at P ≤ 0.05, and tendencies were considered at 0.05 < P < 0.10.
2.3.2. Determinantion of apparent nutrients digestibility For seven consecutive days (days 61–67), apparent nutrients digestibility was measured using faecal bags and complete daily collection of orts and faeces for each animal. After 7 days of collection, all aliquots of daily samples (10% of the total amounts) per animal were bulked, mixed thoroughly, pooled and stored at -20 °C for subsequent analysis. 2.4. Chemical analysis Feed, orts and pooled faecal samples for each animal were thawed and dried at 60 °C for 48 h using a forced-air oven and then ground using Wiley mill grinder. Ground diets and faeces samples were chemically analyzed according to AOAC (2006) for DM (ID number 930.15), OM (ID number 942.05) and CP (as 6.25 × N; ID number 954.01). The neutral detergent fibre was determined according to Van Soest et al. (1991) using an Ankom 200 Fibre Analyzer unit (ANKOM Technology Corporation, Macedon, NY, USA) and expressed exclusive of residual ash (NDFom). Dietary non-fibre carbohydrates (NFC, g/kg) was calculated as 1000 − (NDFom g/kg + CP g/kg + EE g/kg + ash g/ kg). Feed and faecal gross energy (GE) were determined by bomb calorimeter (model C2000 basic IKA–WERKE GmbH and Co. KG, Staufen, Germany). The metabolizable and net energy (ME and NE; Mcal/kg DM) were calculated according to NRC (2001).
3. Results 3.1. In vitro evaluation assay (Experiment 1) Interestingly, net CH4 in ml, ml/kg IVTDOM and ml/kg IVTDDM decreased quadratically (P < 0.05), while IVTDDM and IVTDOM in g/ kg incubated DM were not affected (P > 0.05, Table 3). In addition, the MCP in mg/kg TIVDOM increased quadratically (P = 0.027) in response to increasing DVR levels in the diet. DVR levels had no effect (P > 0.05) on ruminal pH (Table 4). Ruminal NH3-N concentrations increased linearly (P < 0.001) as DVR levels increased in the diet. However, total SCFA, acetate (C2) butyrate (C4), isobutyrate and C2:C3 ratio decreased quadratically (P = 0.036, P = 0.020, P = 0.029, P = 0.046 and P= 0.042 respectively), while propionate proportion (C3) increased quadratically (P = 0.034). In
2.5. Economic viability Economic evaluation aimed to highlight the effectiveness of DVR as an alternative source of protein. The feed cost per kg BWG (US$/kg gain) was calculated as feed cost (US$/head/day) divided by ADG (kg/ day). The net profit was calculated as described by Abo-Zeid et al.
Table 3 Impact of dried vinasse rice (DVR) replacing soybean meal on in vitro gas production (GP), methane production (CH4) and substrate degradability within 24 h of incubation. Item
Net GP, ml Net CH4, ml Net CH4, ml/kg IVTDOM Net CH4, ml/kg IVTDDM IVTDDM, g/kg incubateda IVTDOM, g/kg incubatedb MCP, mg/kg IVTDOM
Replacement levels (g DVR/kg SBM)
SEM
0
250
500
750
159 18.2 29.1 29.5 614 623 273.1
157 17.3 27.4 28.1 613 628 282.4
149 13.7 21.5 21.8 629 627 299.3
152 15.9 25.3 26.1 607 617 274.3
3.58 0.94 1.65 1.66 3.82 3.45 11.19
IVTDDM: in vitro truly degraded dry matter; IVTDOM: in vitro truly degraded organic matter; MCP: microbial crude protein. a Total DM content per bottle was 0.463 g/0.500 g of air-dried substrate. b Total OM content per bottle was 0.453 g/0.500 g of air-dried substrate. 4
Effect (P-value) Linear
Quadratic
0.148 0.329 0.409 0.274 0.452 0.347 0.012
0.336 0.046 0.043 0.046 0.681 0.723 0.027
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Table 4 Impact of dried vinasse rice (DVR) replacing soybean meal on in vitro ruminal fermentation constituents within 24 h of incubation. Item
Replacement levels (g DVR/kg SBM)
pH NH3-N, mg/100ml Short chain fatty acids (SCFA) Total SCFA, mM Acetate, mol/100 mol. Propionate, mol/100 mol. Butyrate, mol/100 mol. Valerate, mol/100 mol. Isobutyrate, mol/100 mol. Isovalerate, mol/100 mol. C2:C3 ratio Protozoa, × 105 cell/ml
SEM
0
250
500
750
6.51 22.3
6.58 17.7
6.52 16.2
6.54 13.3
92.5 43.4 19.4 24.2 1.33 2.47 1.71 2.24 3.97
91.6 43.4 21.9 22.2 1.41 1.40 1.39 1.98 3.01
84.9 39.5 25.4 16.4 1.50 1.09 1.07 1.62 2.65
90.1 42.8 22.3 21.1 1.39 1.21 1.27 1.90 2.19
Effect (P-value) Linear
Quadratic
0.01 1.53
0.603 < 0.001
0.501 0.501
1.39 0.75 1.03 1.35 0.03 0.26 0.11 0.11 0.31
0.954 0.368 0.170 0.537 0.942 0.002 0.125 0.291 0.036
0.036 0.020 0.034 0.029 0.867 0.046 0.183 0.042 0.278
SEM: standard error of the mean; SCFA: short chain fatty acid; C2:C3 ratio: acetate to propionate ratio.
addition, a linear decrease (P = 0.036) in the total protozoal count was observed by increasing DVR levels in the diet.
Table 6 Impact of dried vinasse rice (DVR) replacing soybean meal on intake (g/kg) and apparent digestibility of nutrients (g/kg) of growing Texel lambs.
3.2. Performance evaluation trial (Experiment 2)
Item
Lambs fed on DVR diet showed higher (P < 0.001) BWG, ADG and FE than those fed on control diet after 60 days of the commencement of the feeding trial (Table 5). The DVR diet enhanced the digestibility of CP (P < 0.001) compared to the control diet (Table 6). Lambs fed on the DVR diets had higher DE (P = 0.003), ME (P < 0.001) and NE (P < 0.001) than those fed with control diet. The economic efficiency of the experimental diets is presented in Table 7. The MFP decreased (P < 0.001) with DVR relative to control diets (0.114 vs. 0.118 US$/kg DM). Feed cost (US$/kg gain) was lower when lambs were fed on DVR diet, compared to those fed on control diet (0.97 vs 1.46 US$/kg gain). Replacement of SBM with 500 g DVR /kg increased (P < 0.001) the net profit and economic efficiency relative to control (1.69 vs 1.48 US $/head/day and 1.57 vs 1.04, respectively).
Treatments
Replicates Intake, kg/d Dry matter Organic matter Crude protein Neutral detergent fiber Total apparent digestibility, g/kg Dry matter Organic matter Crude protein Neutral detergent fiber Digestible energy, Mcal/kg DM Metabilizable energy, Mcal/kg DM1 Net energy, Mcal/kg DM1
P-value
Control
DVR
SEM
6
6
ــــــ
ــــــ
1.18 1.12 0.15 0.45
1.15 1.09 0.15 0.43
1.79 1.71 0.28 0.66
0.824 0.787 0.148 0.794
518 543 533b 507 2.01b 1.65b 0.81b
513 543 582a 519 2.83a 2.32a 1.44a
0.05 0.05 0.02 0.13 0.19 0.09 0.08
0.687 0.571 < 0.001 0.150 0.003 < 0.001 < 0.001
a,b
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice. 1 Calculated according to NRC (2001).
4. Discussion 4.1. In vitro evaluation To the best of our knowledge, neither in vitro nor in vivo data to appraise the importance of DVR as an alternative source of protein to SBM has been published yet. Therefore, results comparable to ours are not available for direct evaluations. However, the lack of differences between the effects of different DVR levels on in vitro GP was consistent
Table 7 Impact of dried vinasse rice (DVR) replacing soybean meal on economic viability of experimental diets. Item
Treatments Control
Table 5 Impact of dried vinasse rice (DVR) replacing soybean meal on growth performance of growing Texel lambs. Item
Treatments
MFP, US$/kg DM Feed cost, US$/head/d Feed cost, US$/kg gain Net profit, US$/head/d Economic efficiency
P-value
Control
DVR
SEM
60 6 30.7 36.5 5.80b 0.10b 1.18 0.08b
60 6 30.0 38.1 8.14a 0.14a 1.15 0.12a
ــــــ ــــــ 0.04 0.04 0.01 0.20 1.79 0.01
0.118 0.14 1.46a 1.48b 1.04b
a
P-value DVR 0.114 0.13 0.97b 1.69a 1.57a
SEM b
0.01 0.01 0.14 0.04 0.17
< 0.001 0.224 < 0.001 < 0.001 < 0.001
a,b
Experimental days Replicates IBW, kg FBW, kg BWG, kg/60d ADG, kg/d DMI, kg/d FE, ADG/ DMI
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice; Net profit: (1.5 × ADG) − FCD where 1.5 is market cost of body weight (US$/kg) and FCD is feed cost per day; Economic efficiency: (ADG × 1.5)/(DMI × MFP) where ADG is average daily gain; DMI is the daily dry matter intake (US$/head/d), MFP is market feed price.
ــــــ ــــــ 0.852 0.607 < 0.001 < 0.001 0.824 < 0.001
with the similar lacked effects on IVTDDM and IVTDOM. In comparison to the results reported by Blümmel et al. (1997), the net GP was correlated with digestibility of DM. According to Newbold et al. (1995), ruminal methanogens cohabit with ciliate protozoa to be responsible for 9–25% of methanogenesis. In the present study, dietary inclusion of
a,b
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice; IBW: initial body weight; FBW: final body weight; BWG: body weight gain; DMI: dry matter intake; ADG: average daily gain; FE: feed efficiency (ADG, kg/ DMI, kg). 5
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the most interesting effects were on CH4, MCP, NH3-N and C3 rather than on net GP or feed degradability. It was indicated that mainly ruminal cellulolytic-degrading bacteria utilize branched-SCFA (such as isobutyrate) as carbon skeletons supply for biosynthesis of their branched-chain amino acid (such as valine) and subsequently for enhancing their degradation patterns, enzymatic activities, more growth and proliferation (Andries et al., 1987). The linear decreases in in vitro NH3N concentration and protozoal count were associated with a linear and quadratic decline in isobutyrate (branched-SCFA) which may indicates more microbial growth (Shingfield et al., 2002). It was proved that ruminal protozoa served as butyrate producers (Morgavi et al., 2012), therefore, reduction in C4 was associated with the observed reduction of protozoal count and would lessen the NH3-N concentration (Koenig et al., 2000). However, the pattern of ruminal fermentability of 500 g DVR/kg SBM was distinctly different from other replacement levels.
DVR500 reduced in vitro CH4 formation up to 24% with no adverse effects on feed degradability. Low net CH4 production may likely be a result of high EE content in DVR, which could exert toxic effects on rumen methanogens and protozoal count. It seems that DVR inhibited CH4 formation directly by redirecting H2 toward more C3 production at the expense of C2 and C4 which could be nutritionally advantageous for growing animals. Otherwise, reduction of ruminal CH4 production is indirectly influenced by diminishing ruminal protozoa abundance (Cieslak et al., 2017). Furthermore, low CH4 production could be a result of reduced availability of H2 owing to reduced C2 production via formate formation or depression of protozoa. Production of C2 and C4 associated with more H2 formation which needs to be diverted for production of C3 at the expense of C2 (Wallace et al., 1980). High C3 production suggests that electrons spared from low CH4 production were redirected towards more reduction in fermentation acids (such as C3) as a H2 sink (Moss et al., 2000). Low C2:C3 ratio reflected a shift in C3 production at the expense of C2 the main liver glucogenic precursor (Hammon et al., 2010) and highlighted the positive effects of DVR on the in vitro ruminal fermentation profile. These results are consistent with the results of Huang et al. (1999), who reported that adding 20% dried rice distillers' grain (DRDG) as part of the concentrate depressed C2 and increased C3 and C4 resulting in a decreased C2:C3 ratio. According to Van Soest (1994), ruminal C2 presented an evidence for fiber fermentability, while high ruminal C3 was associated with high fermentation of soluble carbohydrate. The quadratic increase in C3 production in diets containing DVR up to 500 g/kg DM provides a favorable ruminal fermentation pattern responsible evidenced by lowered CH4 production which consequently indicates more energy available for animal growth. Inclusion of DVR in the diet modified the ruminal SCFA profile along with enhancing pyruvate conversion to produce more C3 rather than C2 by consuming H2. Low total SCFA was congruent to that reported by Kleinschmit et al. (2006) when replaced a portion of SBM with corn DDGS in lactating cow diets. Moreover, all current experimental diets had almost equal OM and NDFom contents which may have contributed to the unchanged IVTDOM and consequently to the net GP regardless of DVR increase. Replacement of 500 g DVR/kg SBM resulted in a quadratic reduction in CH4 ml/kg IVTDOM providing more energy supply for SCFA production. The linear decrease in ruminal NH3-N concentrations associated with the quadratic increase in MCP are capable of explaining the enhancements of nitrogen utilization and ruminal bypass of dietary CP for forward absorption in the small intestine. Replacing SBM with up to 500 g DVR/kg diet resulted in the highest quadratic increase in MCP synthesis without adverse effects on feed degradability. However, the magnitude of the response in MCP to replacement of 50% SBM by DVR was more pronounced. This indicates that responses to 750 DVR level may likely be reduced than responses to 500 DVR level that provision a complementary impact by the animal performance. It is likely that, dietary optimization to provide nutrients requirements of growing lambs might require the replacement with 500 g DVR rather than with 250 or 750 g/kg DM. According to Blümmel et al. (1997), in vitro substrate truly degraded (IVTDOM) and GP volume were well positively correlated. Furthermore, truly degraded substrate reveals the amount of feed totally fermented and GP volume indicates the amount of this fermented substrate utilized for SCFA production. In a consequence, the conversion of substrate truly degraded into GP supports the assumptions that degraded substrate was converted to production of SCFA, ruminal fermentative CO2 and CH4. Thus, in vitro GP volume well reflects the fermentability of incubated substrate to SCFA (Blümmel and Ørskov, 1993). The consistent increased MCP in response to DVR levels emphasized that the latter inherently promoted CP utilization by rumen microbes. Similar in vitro results by Li et al. (2012) indicated that greater production of bacterial CP was observed when wheat DDGS replaced portion of barley grain. Compared to the control diet, the individual SCFA proportions were modified by the inclusion of DVR, but
4.2. Performance evaluation Currently, to our knowledge, there is no published data highlighting the effect of DVR, as a source of both protein and energy, on ruminant performance. The current study is the first to evaluate the replacement of SBM with DVR in growing Texel lambs diet. The lack of effect of DVR on DMI was consistent with unchanged apparent digestibility of DM and NDFom. High in vitro C3 could likely be a reflection of the controlled increase in DMI in vivo (Allen et al., 2009) and could contribute to energy deposition as a gluconeogenic precursor for glucose availability and uptake by body tissues. Regardless of the absence of effect on DMI, lambs fed on DVR diet showed higher BWG and ADG than those fed on control diet. It is known that energy is the most limiting factor for MCP synthesis (Owens and Zinn, 1988). Higher BWG and ADG confirm the improved apparent CP digestibility and DE ensuring more production of ruminal fermentable energy, leading to improved MCP synthesis. It seems that combining DVR and SBM (500:500 w/w) was well utilized and considerably effective for increased BWG and FE in growing lambs. From the energetic point of view, the DVR diet possess higher ME and consequently NE for gain which might be attributable to lower CH4 production obtained in the in vitro study. Greater ADG and FE without any change in DMI suggest that DVR partitioned more feed GE into more BWG. It was noted that, higher ruminal C3 likely stimulates the liver oxidation process of acetyl-CoA, elevating the production of ATP (Allen et al., 2009) and generating more energy availability (El-Zaiat et al., 2019). From an energy standpoint, the DVR inclusion was beneficial as evidenced by positive responses in DE, ME and NE digestibility. Additionally, the increase in DE may indicate lower loss of feacal energy when DVR replaced 500 g/ kg DM of dietary SBM. Interestingly, the increase in DE might be associated with the increase in utilization of dietary energetic efficiency with the inclusion of DVR in the diet. These results are supported by high in vitro C3 as DVR levels increased to act as substrates for microbial growth. The increased availability of CP to the animal may explain the greater BWG and better FE observed. However, the responses of ADG and DMI leading to increase of FE with inclusion of DVR in the diet were in contrast with López-Campos et al. (2011) who found a linear reduction in DMI and ADG of lambs fed different amounts of vinasse obtained from the alcoholic fermentation of molasses. Combining SBM with DVR might effectively provide the indispensable amino acids coinciding with enhancement of nutritional value of the diet. More research studies are needed to evaluate this relationship. However, the improved BWG caused by replacing SBM by 500 g DVR /kg does not negate the better utilization of dietary CP and energy as found in the in vitro results. Thus, the use of DVR could result in a better synchrony between the fermented energy and NH3-N utilization by rumen microbes. In our study, replacing 500 g DVR/kg DM for SBM in the diet increased lambs FE probably due to lower energy loss as indicated by the in vitro depression in CH4 production. Replacing 500 g DVR/kg DM for 6
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Conflict of interest statement
SBM reduced CH4 production coupled with lower C2:C3 ratio, since CH4 production is positively proportional to ruminal C2:C3 ratio (Moss et al., 1995). The depression of CH4 production may be attributable to the redirection of H2 metabolism away towards more C3 production, which might be ascribed to help reduce the energy loss, which was probably an explanation for the improved DE and subsequently FE. In our in vitro study, the lack of changes in IVTDDM and IVTDOM until 24 h of incubation could explain similar lacked effect on the in vivo digestibility of DM and OM. According to Castillejos et al. (2006) the reduction in fibre fraction digestibility was associated with lower ruminal C2 proportion. Surprisingly, although reductions in C2 and C4 proportions were observed in the in vitro study, the in vivo NDFom digestibility was not affected. However, the increase in apparent digestibility of CP indicates that dietary inclusion of DVR could enhance rumen microbial growth and activity. Higher apparent CP digestibility accompanied lower in vitro ruminal NH3-N which may be attributed to lower degradation of dietary CP. Therefore, the increased CP digestibility is much associated with the rumen contents of undegradable CP depending on the proportion of the DVR replacing SBM. According to Erasmus et al. (1994), degradability of CP is negatively correlated with amino acids outflow to small intestine. In addition, the DVR seems to be a source of ruminal undegradable CP fraction that coincides with low NH3-N formation and confirms the observed in vitro assay results.The roasted dry DVR may be effective in shifting degradation of CP from the rumen to the intestines without adverse effects on ruminal MCP synthesis which coincided with a decrease in NH3-N concentrations observed in vitro. Dry roasting vinasse rice could efficiently decrease ruminal solubility and degradability of CP and starch enhancing their escape to the stomach and the small intestine (Friedman, 1996) which could compensate the deficiency in amino acids profile in the diet. With respect to economic aspects, lambs fed on DVR diet shared much better BWG, ADG and FE resulting in 14 and 50% higher net profit and economic efficiency than control without compromising ruminal fermentation profile. Including DVR in the diet increased FE and consequently retained more energy resulting in higher BWG presumably due to decreased CH4 production as observed in vitro. The current results are desirable from the applicative and environmental points of view. Redirecting CH4 production towards more BWG is a principal interest of beef producers (Eckard et al., 2010) which form another advantage of DVR over SBM and may support its inclusion in sheep diets. From the applicative point of view, cost-effectiveness of DVR introduce it as a novel alternative source of protein suitable for ruminants diet formation and assures a new solution to animal nutrition challenges, particularly for developing countries. Increased MCP indicated that 50% replacement could alter the quality of CP entering the small intestine compared with the other replacement levels. The current in vitro results inferred that inlusion of DVR in the diet indicates more ruminal nutrients and fermentable energy supply adequate to enhance feed degradability with consequential increase in MCP synthesis. This emphasizes the improvement in lambs BWG and ADG which might be due to the potential complementary effect between DVR and SBM as described previously.
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Direct determination of ammonium in blood and tissue extracts by means of the phenol by chlorite reaction. Clin. Chim. Acta Int. J. Clin.Chem. 8, 5–11. Li, Y., He, M., Li, C., Forster, R., Beauchemin, K.A., Yang, W., 2012. Effects of wheat dried distillers’ grains with soluble and cinnamaldehyde on in vitro fermentation and protein degradation using the Rusitec technique. Arch. Anim. Nutr. 66, 131–148. Longo, C., Bueno, I.C.S., Nozella, E.F., Goddoy, P.B., Cabral Filho, S.L.S., Abdalla, A.L., 2006. The influence of head-space and inoculum dilution on in vitro ruminal methane measurements. In: Soliva, C.R., Takahashi, J., Kreuzer, M. (Eds.), Greenhouse Gases and Animal Agriculture: An Update Int. Congr. Series No. 1293. Elsevier, The Netherlands, pp. 62–65.
5. Conclusions The partial replacement of soybean meal with 500 g DVR /kg had no adverse effects on feed intake and resulted in higher body weight gain, protein digestibility and net profit potentials. All these circumstances should certainly justify the novelty of using DVR which might be an alternative source of protein but also a source of energy in growing Texel lamb diets. However, future studies are needed to elucidate the effects of DVR on rumen microbes and their fermentability characteristics in commercial rations in long-term trials.
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Sciences, Washington, DC, USA, pp. 362. Onodera, R., Henderson, C., 1980. Growth factors of bacteria origin for the culture the rumen oligotrich protozoon Entodinium caudatum. J. Appl. Bacteriol. 8, 125–132. Owens, F.N., Zinn, R., 1988. Protein metabolism of ruminant animal. In: Church, D.C. (Ed.), The Ruminant Animal: Digestive Physiology and Nutrition. Simon and Schuster, Englewood Cliffs, pp. 227–249. Rodrigues, R.C.E., Hu, B., 2017. Vinasse from sugarcane ethanol production: better treatment or better utilization? Front. Energy Res. 5, 7. SAS Institute, 2002. Statistical Analysis System, Version 9.1. SAS Institute Inc., Cary, NC. USA. Shingfield, K.J., Jaakkola, S., Huhtanen, P., 2002. Effect of forage conservation method, concentrate level and propylene glycol on diet digestibility rumen fermentation, blood metabolite concentrations and nutrient utilization of dairy cows. Anim. Feed Sci. Technol. 97 (1), -21. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell Univ. Press, Ithaca, NY. USA, pp. 476. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fibre, neutral detergent fibre, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wallace, R.J., Cheng, K.J., Czerkawski, 1980. Effect of monensin on fermentation characteristics of the artificial rumen. Appl. Environ. Microbiol. 40, 672–674. Zali, A., Eftekhari, M., Fatehi, F., Ganjkhanlou, M., 2017. Effect of vinasse (condensed molasses solubles) on performance and meat chemical composition of Holstein male calves. Ital. J. Anim. Sci. 16 (3), 515–520.
Lópes, M.L., Paulillo, S.C., Godoy, A., Cherubin, R.A., Lorenzi, M.S., Giometti, F.H.C., Bernardino, C.D., Neto, H.B.A., Amorim, H.V., 2016. Ethanol production in Brazil: a bridge between science and industry. Braz. J. Microbiol. 47, 64–76. López-Campos, Ó., Bodas, R., Prieto, N., Frutos, P., Andrés, S., Giráldez, F.J., 2011. Vinasse added to the concentrate for fattening lambs: intake, animal performance, and carcass and meat characteristics. J. Anim. Sci. 89, 1153–1162. Menke, K.H., Raab, L., Salewski, A., Steingass, H., Fritz, D., Schneider, W., 1979. The estimation of the digestibility and metabolizable energy content of ruminant feedstuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric. Sci. 92, 217–222. Morgavi, D.P., Martin, C., Jouany, J.P., Ranilla, M.J., 2012. Rumen protozoa and methanogenesis: not a simple cause-eff ect relationship. Br. J. Nutr. 107, 388–397. Moss, A.R., Givens, D.I., Garnsworthy, P.C., 1995. The effect of supplementing grass silage with barley on digestibility, in sacco degradability, rumen fermentation and methane production in sheep at two levels of intake. Anim. Feed Sci. Technol. 55, 9–33. Moss, A.R., Jouany, J.P., Newbold, J., 2000. Methane production by ruminants: its contribution to global warming. Ann. Zootech. 49, 231–253. Newbold, C.J., Lassalas, B., Jouany, J.P., 1995. The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro. Lett. Appl. Microbiol. 27, 230–234. NRC, 2001. National Research Council, Nutrient Requirements for Dairy Cattle, 7th rev. ed. National Academy Press, Washington, DC, USA, pp. 408. NRC, 2007. National Research Council, Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids, 1sted. National Academy of
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Assessment of using dried vinasse rice to replace soybean meal in lambs diets: In vitro, lambs performance and economic evaluation
T
⁎
Hani M. El-Zaiata, , Diogo D. Réb, Harold O. Patinob, Sobhy M.A. Sallama a
Department of Animal Production, Faculty of Agriculture, University of Alexandria, Aflaton St., El-Shatby, P.O Box 21545, Alexandria, Egypt Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 7712 Bairro Agronomia CEP: 91540000, Porto-Alegre, RS, Brazil
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dried vinasse rice Rumen fermentation Performance Soybean meal Texel lambs
Effects of replacing soybean meal (SBM) with dried vinasse rice (DVR) as an alternative source of protein were evaluated under in vitro and in vivo conditions. Protein from SBM was partially replaced by DVR protein at the levels of zero (DVR0) control, 250 (DVR250), 500 (DVR500) and 750 g (DVR750) of the original concentration. An in vitro semi-automatic system was employed to evaluate gas production, feed degradability and fermentation profile of diets. The ANOVA and regression coefficients indicated that the DVR effective level was 500 g/kg DM. An in vivo experiment on twelve Texel lambs (BW; 30.48 ± 0.45 kg) kept individually in wooden pens and allotted into randomized complete block design with 6 blocks by 2 treatments as: control (diet without DVR) and DVR (500 g/kg DM) lasted for 60 days, and the subsequent 7 days were assigned for the digestibility trial. Upon increasing dietary DVR, net CH4 production decreased quadratically (P < 0.05). Ruminal NH3-N concentration and total protozoal count decreased linearly (P < 0.05) as DVR levels increased. In vitro microbial protein and propionate proportion (C3) increased quadratically (P = 0.027 and P = 0.034, respectively), while total short chain fatty acids, acetate (C2) butyrate, isobutyrate and C2:C3 ratio decreased quadratically (P = 0.036, P = 0.020, P = 0.029, P = 0.046 and P = 0.042, respectively). Lambs fed 500 g DVR/kg DM showed higher (P < 0.001) daily gain, body weight, feed efficiency and crude protein digestibility compared to control. The DVR diet showed higher (P < 0.05) digestible, metabolizable and net energy than control. DVR500 diet reduced (P < 0.001) feed costs and increased (P < 0.001) the net profit compared to control. The use of DVR provides a promising source of both protein and energy for growing lambs with extra potentials for economic advantages without adverse effects on growth performance.
1. Introduction
double productivity (14 ton/ha) have been cultivated and are not used for human consumption but for ethanol production and animal nutrition (EMBRAPA, 2013). Vinasse, a co-product obtained after distillation of fermented materials can be used as a source of nutrients to support ruminant performance (López-Campos et al., 2011; Rodrigues and Hu, 2017). This could allow for more value added to the ethanol production chain and is more environmentally sustainable (Barros et al., 2009). Addition of 13% vinasse to dried sugar beet pulp improves disappearance rates of dry matter with no effect on ruminal pH, ammonia concentration, or individual and total short chain fatty acids (Fernández et al., 2009). Further, inclusion of 5% vinasse in the diet showed positive effects on nitrogen supply and modified positively body composition of growing calves (Zali et al., 2017). Feeding 100 and 200 g of vinasse/kg of concentrate mixture increased ruminal pH and reduced feed intake while increased the feed to gain ratio in fattening lambs
Soybean meal (SBM) represents the most frequently used source of dietary protein in livestock feeding, however, it is one of the most expensive ingredients. Sheep owners in the developing countries are not always able to supplement their lambs concentrate mixtures with SBM, though it is necessary to meet the nutritional demands of lambs during the early growth phase. Therefore, looking for low price protein source as an alternative to SBM, while keeping reasonable nutritive value to reduce dietary cost and increase economic profits is a must. New technologies to produce ethanol from different plants raw materials such as sugarcane, corn and other feedstocks are available and applicable in Brazil (Lópes et al., 2016). Meanwhile, challenges facing biofuel production chain seek an efficient methodology to get rid of the generated co-products (Zali et al., 2017). New rice cultivars with
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Corresponding author. E-mail addresses: [email protected], [email protected] (H.M. El-Zaiat).
https://doi.org/10.1016/j.smallrumres.2019.01.003 Received 11 July 2018; Received in revised form 17 November 2018; Accepted 14 January 2019 Available online 14 February 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
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ground corn, SBM and minerals were formulated for in vitro assay evaluation. The control diet contained 36 g SBM/kg DM as the main source of protein and was free of DVR (DVR0). In the remaining three DVR diets, protein from SBM was partially replaced by DVR protein at the levels of 250 (DVR250), 500 (DVR500), and 750 g (DVR750) of the original SBM amount which corresponded to 0, 13, 26, and 39 g of DVR, respectively, of the dietary DM. The feed ingredients and chemical composition of the experimental diets are presented in Table 2. The readings of GP pressure in the bottles headspace were taken using semi-automatic system procedure equipped with a pressure transducer and a data logger (Pressure Press Data GN200, Sao Paulo, Brazil) according to Bueno et al. (2005). To avoid the effects of unusual ruminal conditions, six adult rumencannulated Barki sheep (BW, 49.0 ± 2.3 kg) were used as rumen content donors. Animals were fed Berseem (clover) hay ad libitum plus 0.750 kg/100 kg BW of commercial concentrate mixture (145 g CP/kg DM) plus mineral premix to meet nutrients requirement (NRC, 2007). Ruminal liquid and solid fractions (500:500 v/v) were collected separately from each animal before morning feeding and placed separately into thermo containers (preheated with water at 39ºC) under anaerobic conditions and transported immediately to the laboratory. The two fractions were blended at 1:1 (v/v) for 10 s, strained by hand through four layers of cheesecloth before combining with buffer solution, then held under CO2 to ensure anaerobic conditions in a water bath (39ºC) until used (Bueno et al., 2005). Each treatment was incubated in three inocula, each prepared from two animals. This was necessary due to the large amount of inoculum needed and to ensure that the natural variation among animals was maintained and at the same time, the effects of unusual rumen inocula or rumen environments were reduced. For each inoculum, four bottles per treatment were prepared: two for in vitro truly degraded organic matter (IVTDOM) and in vitro truly degraded dry matter (IVTDDM) determination and the other two bottles for determining fermentation constituents. A corresponding four bottles consisted only of rumen fluid and buffer solution without substrate (blank) to correct the GP from the inoculum, and further, four bottles contained Berseem hay as an internal standard to correct the variation between inoculum. The total bottles of the procedure were: 72 bottles ((4 replicates × 4 treatments+ 4 internal standard + 4 blanks) × 3 inoculum) for all inocula. A representative substrate sample was ovendried and ground in a Wiley mill to pass through a 1 mm screen for use in the incubations assay. A 300 mg of dried substrate were accurately weighed into 120 mL glass bottles and stored overnight at 39ºC. Each bottle (pre-warmed to 39ºC) was then injected with 30 mL of MB9 medium (Onodera and Henderson, 1980) and 15 ml of rumen inoculum. The pH was adjusted to 6.8 and CO2 was flushed for 30 min. After filling, bottles were then closed with a rubber stoppers (Bellco Glass Inc, Vineland, NJ, USA), manually shaken to mix and placed in a forced air incubator at 39ºC (FALC instruments S.R.L., Treviglio, Italy) for 24 h. The gas headspace pressure (75 mL) was recorded at distinct incubation times 6, 12 and 24 h from the beginning of incubation using a pressure transducer. The gas volume (mL) = 4.974 × measured pressure (psi) + 0.171 (n = 500; r2 = 0.99; data are not reported). For the CH4 analysis, representative gas samples of 2 mL were collected from the bottles at distinct incubation times using 5 mL syringe (med Dawliaico, Assiut, Egypt) and accumulated in 10 mL Vacutainer tubes (BD Vacutainer® Tubes, NJ, USA) to determine the CH4 concentration. After each gas sampling, the bottles were vented to release the gas pressure accumulation, shaken manually and immediately returned to the incubator oven thereafter. The CH4 concentration was determined by gas chromatography (Model 7890, Agilent Technologies, Inc, Colorado 80537, USA) with three valve system using 1/8 inch packed columns having early back flush of the C6 components and equipped with a thermal conductivity detector. Separation was achieved using micro packed column using helium as carrier gas with a flow rate of 28.0 mL/min. The detector and
(López-Campos et al., 2011). The limitation of exploiting vinasse in animal feeds is associated with its high-water contents which make it difficult to handle, transport and store. Historically, several studies have been conducted to evaluate different types of wet vinasse, but no definitive results were accomplished for the utilization of dry vinasse as a co-product obtained from rice fermentation in animal feeding. We hypothesized that inclusion of dried vinasse obtained from the alcoholic fermentation of rice (DVR) can improve the dietary nutritive value, digestibility and animal performance. Therefore, the objectives of this study were initiated to evaluate the effect of DVR as an alternative source of protein on in vitro ruminal fermentation, in vivo apparent digestibility, growth performance and feed costs of Texel sheep. 2. Materials and methods The in vitro evaluation assays were conducted at Laboratory of Animal Nutrition, Department of Animal Production, Faculty of Agriculture, Alexandria University, Egypt. The in vivo experiment was carried out at ruminant sector of Laboratório de Ensino Zootécnico, Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul (UFRGS), Brasil (30°_04_21_21 S 51°_08_13_88 N). All experimental procedures involving the use of animals were approved by ethical committee for care and use of agricultural animals in agricultural research and teaching experiments obtained from UFRGS, Brazil. 2.1. Preparations of dried vinasse rice (DVR) The vinasse co-product was obtained from the fermentation process of rice grains. After removing the hulls rice grains are ground to 400 μm, saccharified, fermented, distilled with alcohol plates in a distillation column system and the vinasse is separated, thereafter, dried in a forced-air oven at 40 °C for 72 h. The mineral composition and characteristics of DVR are presented in Table 1. 2.2. In vitro gas production (GP) evaluation assay (Experiment 1) In vitro batch culture assay (Experiment 1) was employed to evaluate the effects of replacing dietary soybean meal (SBM) with increasing levels of dried vinasse rice (DVR) incubated at 39 °C for 24 h and to select the effective level of DVR on nutrients digestibility as well as lambs performance (Experiment 2). The experimental diet used as a fermentation substrate was formulated (a mixture of forage and concentrate 75:25, w/w) as presented in Table 2. The basal experimental diet (127 g CP /kg of DM) contained 750 g/kg DM chopped Oats (Avena Strigosa) hay and 250 g/kg DM concentrate mixture consisting of Table 1 Minerals composition and characterization of experimental dried vinasse rice (DVR). Items characterization pH Organic carbon, g/kg Nitrogen, g/kg DM Gross energy, Mcal/kg DM Minerals, g/kg DM Phosphorus Potassium Calcium Sodium Magnesium Sulfur Trace elements Copper Zinc Manganese
Quantity
3.8 470 48 5.12 7.10 5.00 0.80 0.43 2.50 3.70 0.018 0.070 0.064
2
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Table 2 Feed ingredients and chemical composition of experimental diets on DM basis. Items
Ingredients Oats hay Maize grains Soybean meal (SBM) Dried vinasse rice (DVR) Limestone Mineral and vitamin mixturesa GE, Mcal/kg DM ME, Mcal/kg DMb NEm, Mcal/kg DMb NEg, Mcal/kg DMb Chemical composition, g/kg DM OM CP EE NFCc NDFom
Oats hay
SBM
DVR
Maize grain
Replacement levels (g DVR/kg SBM) 0
250
500
750
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ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ ــــــ
750 210 36 0 1.50 2.50 3.38 2.77 1.84 0.45
750 206 27 13 1.50 2.50 3.45 2.83 1.89 0.49
750 202 18 26 1.50 2.50 3.51 2.88 1.93 0.53
750 198 9 39 1.50 2.50 3.60 2.95 1.99 0.58
842 119 26 244 452
936 441 34 352 154
960 301 155 453 123
987 107 37 817 165
872 128 28 337 379
873 127 30 331 379
873 127 32 331 378
873 127 33 332 378
a Composition: Each one kg consisting of Mn, 75 mg; Zn, 70 mg; Se, 300; Fe, 50 mg; Cu, 8 mg; I, 750 mg; Vit A, 8000 IU; Vit D3, 2000 ICU; Vit K3, 1.8 IU; Vit B1, 1.8 mg; Vit B2, 6 mg; Vit B12, 12 mg; Vit B6, 2.8 mg; thiamine, 3 mg; folic acid, 1.0 mg; Biotin, 60 mg; Pantothenic acid, 10 mg; nicotinic acid and 40 mg. DVR: dried vinasse rice; GE: gross energy; ME: metabolizable energy; NEm: net energy for maintenance; NEg: net energy for gain; DM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; NDFom: neutral detergent fibre expressed exclusive of residual ash. b Calculated according to NRC (2001). c NFC: non-fibre carbohydrates (g/kg) = 1000 − (NDFom g/kg + CP g/kg + EE g/kg + ash g/kg).
described by Konitzer and Voigt, (1963). Aliquots of 2 ml subsample of ruminal fluid were mixed with 4 ml of methyl green formalin-saline solution, preserved in glass bottle and stored in darkness at room temperature until total protozoa enumeration (El-Zaiat et al., 2018). For protozoa counting, bottles were mixed well immediately before counting and about 10 μl of the sample were loaded onto a counting chamber (Neubauer Improved Bright-Line counting cell, 0.1 mm depth; Labor Optik, Lancing, UK) and held on an optical microscope with an objective 45x/.66 according to the procedure described by Dehority (1993). The short chain fatty acids (SCFA) concentration was determined using GC as described by Abo-Zeid et al. (2017) using a gas chromatograph (Thermo Fisher Scientific, Inc., TRACE1300, Rodano, Milan, Italy) fitted with an AS3800 autosampler and equipped with a capillary column FFAP (260 N 225 P; 0.5 μm film o.d., 0.53 μm i.d., and 30 m length; J and W Agilent Technologies Inc., Palo Alto, CA). Nitrogen at 7 mL min−1 was used as carrier gas. Air, hydrogen and nitrogen fluxes (make up gas) were kept at 450, 40, and 35 mL/min, respectively. A 0.1 μL aliquot was injected in splitless mode for the entire run with 31.35 mL min−1 of H2 flux (63.432 Pa). Injector and flame ionization detector (FID) temperatures were held isothermally at 250 °C. Oven heating slope was 80 °C (1 min), 120 °C (20 °C min−1 for 3 min), and 205 °C (10 °C min−1 for 2 min), with 9 min overall analytical time.
column temperatures were 250 °C and 60 °C, respectively. The test of linearity and calibration were accomplished using a standard gas curve in the range of probable concentrations of the samples. The produced CH4 was calculated according to the method of Longo et al. (2006) as follows: CH4, ml = (Total GP, ml + Headspace, 75 ml) × CH4 concentration (ml/ml). The net GP and CH4 values were determined by correcting for the corresponding blank values.
2.2.1. In vitro degradablity and fermentation constituents After 24 h of incubation, the fermentation process was inhibited by swirling all bottles in ice. For determining IVTDOM and IVTDDM, bottles were injected with 70 ml of neutral detergent solution immediately and incubated at 105ºC for 3 h, assuming complete removal of microbial cells by the detergent as described by Blümmel and Becker (1997). Bottles contents were filtered, washed with hot water (50 °C) and then acetone and transferred into pre-weighed crucibles, and residual DM was estimated. The residual DM left above was presented as IVTDDM and calculated as following: = {(g Sample DM – g Residue DM − g Blank DM)/g incubated substrate}*1000. The dried residual was then ashed at 550 °C to calculate IVTDOM as following: = {(g Sample OM – g Residue OM − g Blank OM)/g incubated substrate}*1000
2.3. Animals and experimental design (Experiment 2) Twelve castrated Texel crossbred lambs (age 12 months, initial body weight, IBW 30.48 ± 0.45 kg) individually housed in wooden pens were allotted into completely randomized block design with 6 blocks and 2 dietary treatments; control (basal diet without DVR) and DVR (500 g/kg DM). The experiment lasted for 60 days to evaluate lamb performance, whereas the subsequent 7 days were assigned for the nutrient digestibility trial. The pens were equipped with feeders and water buckets for drinking. Two weeks before the onset of the experiment, lambs were treated for internal parasites with oral administrations of levamisole chloridate (Ripercol; Fort Dodge Saúde Animal® LTDA, Campinas, SP, Brazil) as anthelmintic according to BW.
The microbial crude protein (MCP) was calculated according to Menke et al. (1979) as: MCP (mg/kg TDOM) = TDOM, g/kg incubated – (net GP, mL × 2.2) where 2.2 (mg/mL) is the stoichiometric factor which expresses organic matter (mg) required for SCFA gas combined with one mL of GP (Blümmel et al., 1997). The liquid phase of the other two bottles was used for the fermentation constituents and protozoal count assessments. Ruminal fluid pH was measured immediately after collection using a portable pH meter (GLP 21 model; CRISON, Barcelona, Spain). The ammonia (NH3-N) concentrations were determined colorimetrically by spectrophotometer (Alpha-1101 model; Labnics Equipment, California, USA) using commercial lab test (SPINREACT, Ctra. Santa Coloma, 7, Girona, Spain) as 3
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(2017) using the following formula = (market price of BW × ADG) − FCD where the market cost of BW is 1.5 US$/kg and FCD is the feed cost per day. The market feed price (MFP) of oat hay, maize grain, SBM and DVR were 0.10, 0.15, 0.33 and 0.09 US$/kg respectively. Economic efficiency was calculated as output/input: (ADG × 1.5)/(DMI × MFP) to elucidate the practical efficiency of DVR on animal performance.
2.3.1. Performance and feeding management The total mixed ration (TMR) diet consisted of 750 g/kg chopped oats (Avena Strigosa) hay and 250 g/kg concentrate (75:25 forage:concentrate, F:C). Lambs were fed TMR diet twice a day at 0900 and 1700 h in equal portions allowing extra refusal amount of approximately 10% of the daily DMI to meet their nutritional requirements (NRC, 2007). Lambs were allowed feed ad libitum with free access to fresh water throughout the experiment. The amounts of feed offered and refused were recorded daily to estimate feed dry matter intake (DMI) as the difference between daily feed supply and residues. Weekly feed samples were collected to adjust dietary F:C ratio and to be kept for subsequent analyses. Animals were weighed every 30 days before the morning feeding after 16 h fasting in order to monitor body weight changes. Body weight gain (BWG) was calculated as the difference between final body weight (FBW) and IBW; average daily gain (ADG) was the total BWG divided by 60-days; feed efficiency (FE) was obtained by dividing ADG by DMI.
2.6. Statistical analysis In the in vitro assay, each experimental treatment was incubated in duplicate (analytical replicates) and completed in one run for all treatments, using three inocula (statistical replicates) (6 values per treatment). Prior to statistical analysis, in vitro analytical replicates data from the different bottles were averaged. Data from in vitro experiment were analyzed using the PROC MIXED procedure of SAS (version 9.1, SAS Inst. Inc., 2002, Cary, NC). The statistical model included the fixed effect of diet DVR levels (i = DVR0, DVR250, DVR500 and DVR750). In vitro assay data were subjected to one-way ANOVA and regression to test the effects of incremental levels of DVR for SBM on the measured variables. However, because there was no cubic effect, only the DVR linear and quadratic effects are discussed. Growth performance data were analyzed using the MIXED procedure for a randomized complete block design according to the model: Yijk = μ + Ti + Pj + Bk + PTij + еijk where Yijk = observations mean, μ = overall mean, Ti = fixed effect of DVR levels (i = DVR0 and DVR500), Pj= fixed effect of period (j = 1 or 4), Bk = random effect of block (k = 1 to 6), PTij= interaction of DVR × period and eijk = residual error. The lamb nutrients digestibility and economic efficiency were analysed excluding the random effect of block: using the following statistical model as Yi = μ + Ti + ei, where Yi = observations mean, μ = overall mean, Tj = treatment effect (j = 1 to 2), and ei = residual error. Statistical significance level was declared at P ≤ 0.05, and tendencies were considered at 0.05 < P < 0.10.
2.3.2. Determinantion of apparent nutrients digestibility For seven consecutive days (days 61–67), apparent nutrients digestibility was measured using faecal bags and complete daily collection of orts and faeces for each animal. After 7 days of collection, all aliquots of daily samples (10% of the total amounts) per animal were bulked, mixed thoroughly, pooled and stored at -20 °C for subsequent analysis. 2.4. Chemical analysis Feed, orts and pooled faecal samples for each animal were thawed and dried at 60 °C for 48 h using a forced-air oven and then ground using Wiley mill grinder. Ground diets and faeces samples were chemically analyzed according to AOAC (2006) for DM (ID number 930.15), OM (ID number 942.05) and CP (as 6.25 × N; ID number 954.01). The neutral detergent fibre was determined according to Van Soest et al. (1991) using an Ankom 200 Fibre Analyzer unit (ANKOM Technology Corporation, Macedon, NY, USA) and expressed exclusive of residual ash (NDFom). Dietary non-fibre carbohydrates (NFC, g/kg) was calculated as 1000 − (NDFom g/kg + CP g/kg + EE g/kg + ash g/ kg). Feed and faecal gross energy (GE) were determined by bomb calorimeter (model C2000 basic IKA–WERKE GmbH and Co. KG, Staufen, Germany). The metabolizable and net energy (ME and NE; Mcal/kg DM) were calculated according to NRC (2001).
3. Results 3.1. In vitro evaluation assay (Experiment 1) Interestingly, net CH4 in ml, ml/kg IVTDOM and ml/kg IVTDDM decreased quadratically (P < 0.05), while IVTDDM and IVTDOM in g/ kg incubated DM were not affected (P > 0.05, Table 3). In addition, the MCP in mg/kg TIVDOM increased quadratically (P = 0.027) in response to increasing DVR levels in the diet. DVR levels had no effect (P > 0.05) on ruminal pH (Table 4). Ruminal NH3-N concentrations increased linearly (P < 0.001) as DVR levels increased in the diet. However, total SCFA, acetate (C2) butyrate (C4), isobutyrate and C2:C3 ratio decreased quadratically (P = 0.036, P = 0.020, P = 0.029, P = 0.046 and P= 0.042 respectively), while propionate proportion (C3) increased quadratically (P = 0.034). In
2.5. Economic viability Economic evaluation aimed to highlight the effectiveness of DVR as an alternative source of protein. The feed cost per kg BWG (US$/kg gain) was calculated as feed cost (US$/head/day) divided by ADG (kg/ day). The net profit was calculated as described by Abo-Zeid et al.
Table 3 Impact of dried vinasse rice (DVR) replacing soybean meal on in vitro gas production (GP), methane production (CH4) and substrate degradability within 24 h of incubation. Item
Net GP, ml Net CH4, ml Net CH4, ml/kg IVTDOM Net CH4, ml/kg IVTDDM IVTDDM, g/kg incubateda IVTDOM, g/kg incubatedb MCP, mg/kg IVTDOM
Replacement levels (g DVR/kg SBM)
SEM
0
250
500
750
159 18.2 29.1 29.5 614 623 273.1
157 17.3 27.4 28.1 613 628 282.4
149 13.7 21.5 21.8 629 627 299.3
152 15.9 25.3 26.1 607 617 274.3
3.58 0.94 1.65 1.66 3.82 3.45 11.19
IVTDDM: in vitro truly degraded dry matter; IVTDOM: in vitro truly degraded organic matter; MCP: microbial crude protein. a Total DM content per bottle was 0.463 g/0.500 g of air-dried substrate. b Total OM content per bottle was 0.453 g/0.500 g of air-dried substrate. 4
Effect (P-value) Linear
Quadratic
0.148 0.329 0.409 0.274 0.452 0.347 0.012
0.336 0.046 0.043 0.046 0.681 0.723 0.027
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Table 4 Impact of dried vinasse rice (DVR) replacing soybean meal on in vitro ruminal fermentation constituents within 24 h of incubation. Item
Replacement levels (g DVR/kg SBM)
pH NH3-N, mg/100ml Short chain fatty acids (SCFA) Total SCFA, mM Acetate, mol/100 mol. Propionate, mol/100 mol. Butyrate, mol/100 mol. Valerate, mol/100 mol. Isobutyrate, mol/100 mol. Isovalerate, mol/100 mol. C2:C3 ratio Protozoa, × 105 cell/ml
SEM
0
250
500
750
6.51 22.3
6.58 17.7
6.52 16.2
6.54 13.3
92.5 43.4 19.4 24.2 1.33 2.47 1.71 2.24 3.97
91.6 43.4 21.9 22.2 1.41 1.40 1.39 1.98 3.01
84.9 39.5 25.4 16.4 1.50 1.09 1.07 1.62 2.65
90.1 42.8 22.3 21.1 1.39 1.21 1.27 1.90 2.19
Effect (P-value) Linear
Quadratic
0.01 1.53
0.603 < 0.001
0.501 0.501
1.39 0.75 1.03 1.35 0.03 0.26 0.11 0.11 0.31
0.954 0.368 0.170 0.537 0.942 0.002 0.125 0.291 0.036
0.036 0.020 0.034 0.029 0.867 0.046 0.183 0.042 0.278
SEM: standard error of the mean; SCFA: short chain fatty acid; C2:C3 ratio: acetate to propionate ratio.
addition, a linear decrease (P = 0.036) in the total protozoal count was observed by increasing DVR levels in the diet.
Table 6 Impact of dried vinasse rice (DVR) replacing soybean meal on intake (g/kg) and apparent digestibility of nutrients (g/kg) of growing Texel lambs.
3.2. Performance evaluation trial (Experiment 2)
Item
Lambs fed on DVR diet showed higher (P < 0.001) BWG, ADG and FE than those fed on control diet after 60 days of the commencement of the feeding trial (Table 5). The DVR diet enhanced the digestibility of CP (P < 0.001) compared to the control diet (Table 6). Lambs fed on the DVR diets had higher DE (P = 0.003), ME (P < 0.001) and NE (P < 0.001) than those fed with control diet. The economic efficiency of the experimental diets is presented in Table 7. The MFP decreased (P < 0.001) with DVR relative to control diets (0.114 vs. 0.118 US$/kg DM). Feed cost (US$/kg gain) was lower when lambs were fed on DVR diet, compared to those fed on control diet (0.97 vs 1.46 US$/kg gain). Replacement of SBM with 500 g DVR /kg increased (P < 0.001) the net profit and economic efficiency relative to control (1.69 vs 1.48 US $/head/day and 1.57 vs 1.04, respectively).
Treatments
Replicates Intake, kg/d Dry matter Organic matter Crude protein Neutral detergent fiber Total apparent digestibility, g/kg Dry matter Organic matter Crude protein Neutral detergent fiber Digestible energy, Mcal/kg DM Metabilizable energy, Mcal/kg DM1 Net energy, Mcal/kg DM1
P-value
Control
DVR
SEM
6
6
ــــــ
ــــــ
1.18 1.12 0.15 0.45
1.15 1.09 0.15 0.43
1.79 1.71 0.28 0.66
0.824 0.787 0.148 0.794
518 543 533b 507 2.01b 1.65b 0.81b
513 543 582a 519 2.83a 2.32a 1.44a
0.05 0.05 0.02 0.13 0.19 0.09 0.08
0.687 0.571 < 0.001 0.150 0.003 < 0.001 < 0.001
a,b
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice. 1 Calculated according to NRC (2001).
4. Discussion 4.1. In vitro evaluation To the best of our knowledge, neither in vitro nor in vivo data to appraise the importance of DVR as an alternative source of protein to SBM has been published yet. Therefore, results comparable to ours are not available for direct evaluations. However, the lack of differences between the effects of different DVR levels on in vitro GP was consistent
Table 7 Impact of dried vinasse rice (DVR) replacing soybean meal on economic viability of experimental diets. Item
Treatments Control
Table 5 Impact of dried vinasse rice (DVR) replacing soybean meal on growth performance of growing Texel lambs. Item
Treatments
MFP, US$/kg DM Feed cost, US$/head/d Feed cost, US$/kg gain Net profit, US$/head/d Economic efficiency
P-value
Control
DVR
SEM
60 6 30.7 36.5 5.80b 0.10b 1.18 0.08b
60 6 30.0 38.1 8.14a 0.14a 1.15 0.12a
ــــــ ــــــ 0.04 0.04 0.01 0.20 1.79 0.01
0.118 0.14 1.46a 1.48b 1.04b
a
P-value DVR 0.114 0.13 0.97b 1.69a 1.57a
SEM b
0.01 0.01 0.14 0.04 0.17
< 0.001 0.224 < 0.001 < 0.001 < 0.001
a,b
Experimental days Replicates IBW, kg FBW, kg BWG, kg/60d ADG, kg/d DMI, kg/d FE, ADG/ DMI
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice; Net profit: (1.5 × ADG) − FCD where 1.5 is market cost of body weight (US$/kg) and FCD is feed cost per day; Economic efficiency: (ADG × 1.5)/(DMI × MFP) where ADG is average daily gain; DMI is the daily dry matter intake (US$/head/d), MFP is market feed price.
ــــــ ــــــ 0.852 0.607 < 0.001 < 0.001 0.824 < 0.001
with the similar lacked effects on IVTDDM and IVTDOM. In comparison to the results reported by Blümmel et al. (1997), the net GP was correlated with digestibility of DM. According to Newbold et al. (1995), ruminal methanogens cohabit with ciliate protozoa to be responsible for 9–25% of methanogenesis. In the present study, dietary inclusion of
a,b
Means in the same row with different superscripts are significantly differ (P < 0.05). SEM: standard error of the mean; DVR: dried vinasse rice; IBW: initial body weight; FBW: final body weight; BWG: body weight gain; DMI: dry matter intake; ADG: average daily gain; FE: feed efficiency (ADG, kg/ DMI, kg). 5
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the most interesting effects were on CH4, MCP, NH3-N and C3 rather than on net GP or feed degradability. It was indicated that mainly ruminal cellulolytic-degrading bacteria utilize branched-SCFA (such as isobutyrate) as carbon skeletons supply for biosynthesis of their branched-chain amino acid (such as valine) and subsequently for enhancing their degradation patterns, enzymatic activities, more growth and proliferation (Andries et al., 1987). The linear decreases in in vitro NH3N concentration and protozoal count were associated with a linear and quadratic decline in isobutyrate (branched-SCFA) which may indicates more microbial growth (Shingfield et al., 2002). It was proved that ruminal protozoa served as butyrate producers (Morgavi et al., 2012), therefore, reduction in C4 was associated with the observed reduction of protozoal count and would lessen the NH3-N concentration (Koenig et al., 2000). However, the pattern of ruminal fermentability of 500 g DVR/kg SBM was distinctly different from other replacement levels.
DVR500 reduced in vitro CH4 formation up to 24% with no adverse effects on feed degradability. Low net CH4 production may likely be a result of high EE content in DVR, which could exert toxic effects on rumen methanogens and protozoal count. It seems that DVR inhibited CH4 formation directly by redirecting H2 toward more C3 production at the expense of C2 and C4 which could be nutritionally advantageous for growing animals. Otherwise, reduction of ruminal CH4 production is indirectly influenced by diminishing ruminal protozoa abundance (Cieslak et al., 2017). Furthermore, low CH4 production could be a result of reduced availability of H2 owing to reduced C2 production via formate formation or depression of protozoa. Production of C2 and C4 associated with more H2 formation which needs to be diverted for production of C3 at the expense of C2 (Wallace et al., 1980). High C3 production suggests that electrons spared from low CH4 production were redirected towards more reduction in fermentation acids (such as C3) as a H2 sink (Moss et al., 2000). Low C2:C3 ratio reflected a shift in C3 production at the expense of C2 the main liver glucogenic precursor (Hammon et al., 2010) and highlighted the positive effects of DVR on the in vitro ruminal fermentation profile. These results are consistent with the results of Huang et al. (1999), who reported that adding 20% dried rice distillers' grain (DRDG) as part of the concentrate depressed C2 and increased C3 and C4 resulting in a decreased C2:C3 ratio. According to Van Soest (1994), ruminal C2 presented an evidence for fiber fermentability, while high ruminal C3 was associated with high fermentation of soluble carbohydrate. The quadratic increase in C3 production in diets containing DVR up to 500 g/kg DM provides a favorable ruminal fermentation pattern responsible evidenced by lowered CH4 production which consequently indicates more energy available for animal growth. Inclusion of DVR in the diet modified the ruminal SCFA profile along with enhancing pyruvate conversion to produce more C3 rather than C2 by consuming H2. Low total SCFA was congruent to that reported by Kleinschmit et al. (2006) when replaced a portion of SBM with corn DDGS in lactating cow diets. Moreover, all current experimental diets had almost equal OM and NDFom contents which may have contributed to the unchanged IVTDOM and consequently to the net GP regardless of DVR increase. Replacement of 500 g DVR/kg SBM resulted in a quadratic reduction in CH4 ml/kg IVTDOM providing more energy supply for SCFA production. The linear decrease in ruminal NH3-N concentrations associated with the quadratic increase in MCP are capable of explaining the enhancements of nitrogen utilization and ruminal bypass of dietary CP for forward absorption in the small intestine. Replacing SBM with up to 500 g DVR/kg diet resulted in the highest quadratic increase in MCP synthesis without adverse effects on feed degradability. However, the magnitude of the response in MCP to replacement of 50% SBM by DVR was more pronounced. This indicates that responses to 750 DVR level may likely be reduced than responses to 500 DVR level that provision a complementary impact by the animal performance. It is likely that, dietary optimization to provide nutrients requirements of growing lambs might require the replacement with 500 g DVR rather than with 250 or 750 g/kg DM. According to Blümmel et al. (1997), in vitro substrate truly degraded (IVTDOM) and GP volume were well positively correlated. Furthermore, truly degraded substrate reveals the amount of feed totally fermented and GP volume indicates the amount of this fermented substrate utilized for SCFA production. In a consequence, the conversion of substrate truly degraded into GP supports the assumptions that degraded substrate was converted to production of SCFA, ruminal fermentative CO2 and CH4. Thus, in vitro GP volume well reflects the fermentability of incubated substrate to SCFA (Blümmel and Ørskov, 1993). The consistent increased MCP in response to DVR levels emphasized that the latter inherently promoted CP utilization by rumen microbes. Similar in vitro results by Li et al. (2012) indicated that greater production of bacterial CP was observed when wheat DDGS replaced portion of barley grain. Compared to the control diet, the individual SCFA proportions were modified by the inclusion of DVR, but
4.2. Performance evaluation Currently, to our knowledge, there is no published data highlighting the effect of DVR, as a source of both protein and energy, on ruminant performance. The current study is the first to evaluate the replacement of SBM with DVR in growing Texel lambs diet. The lack of effect of DVR on DMI was consistent with unchanged apparent digestibility of DM and NDFom. High in vitro C3 could likely be a reflection of the controlled increase in DMI in vivo (Allen et al., 2009) and could contribute to energy deposition as a gluconeogenic precursor for glucose availability and uptake by body tissues. Regardless of the absence of effect on DMI, lambs fed on DVR diet showed higher BWG and ADG than those fed on control diet. It is known that energy is the most limiting factor for MCP synthesis (Owens and Zinn, 1988). Higher BWG and ADG confirm the improved apparent CP digestibility and DE ensuring more production of ruminal fermentable energy, leading to improved MCP synthesis. It seems that combining DVR and SBM (500:500 w/w) was well utilized and considerably effective for increased BWG and FE in growing lambs. From the energetic point of view, the DVR diet possess higher ME and consequently NE for gain which might be attributable to lower CH4 production obtained in the in vitro study. Greater ADG and FE without any change in DMI suggest that DVR partitioned more feed GE into more BWG. It was noted that, higher ruminal C3 likely stimulates the liver oxidation process of acetyl-CoA, elevating the production of ATP (Allen et al., 2009) and generating more energy availability (El-Zaiat et al., 2019). From an energy standpoint, the DVR inclusion was beneficial as evidenced by positive responses in DE, ME and NE digestibility. Additionally, the increase in DE may indicate lower loss of feacal energy when DVR replaced 500 g/ kg DM of dietary SBM. Interestingly, the increase in DE might be associated with the increase in utilization of dietary energetic efficiency with the inclusion of DVR in the diet. These results are supported by high in vitro C3 as DVR levels increased to act as substrates for microbial growth. The increased availability of CP to the animal may explain the greater BWG and better FE observed. However, the responses of ADG and DMI leading to increase of FE with inclusion of DVR in the diet were in contrast with López-Campos et al. (2011) who found a linear reduction in DMI and ADG of lambs fed different amounts of vinasse obtained from the alcoholic fermentation of molasses. Combining SBM with DVR might effectively provide the indispensable amino acids coinciding with enhancement of nutritional value of the diet. More research studies are needed to evaluate this relationship. However, the improved BWG caused by replacing SBM by 500 g DVR /kg does not negate the better utilization of dietary CP and energy as found in the in vitro results. Thus, the use of DVR could result in a better synchrony between the fermented energy and NH3-N utilization by rumen microbes. In our study, replacing 500 g DVR/kg DM for SBM in the diet increased lambs FE probably due to lower energy loss as indicated by the in vitro depression in CH4 production. Replacing 500 g DVR/kg DM for 6
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Conflict of interest statement
SBM reduced CH4 production coupled with lower C2:C3 ratio, since CH4 production is positively proportional to ruminal C2:C3 ratio (Moss et al., 1995). The depression of CH4 production may be attributable to the redirection of H2 metabolism away towards more C3 production, which might be ascribed to help reduce the energy loss, which was probably an explanation for the improved DE and subsequently FE. In our in vitro study, the lack of changes in IVTDDM and IVTDOM until 24 h of incubation could explain similar lacked effect on the in vivo digestibility of DM and OM. According to Castillejos et al. (2006) the reduction in fibre fraction digestibility was associated with lower ruminal C2 proportion. Surprisingly, although reductions in C2 and C4 proportions were observed in the in vitro study, the in vivo NDFom digestibility was not affected. However, the increase in apparent digestibility of CP indicates that dietary inclusion of DVR could enhance rumen microbial growth and activity. Higher apparent CP digestibility accompanied lower in vitro ruminal NH3-N which may be attributed to lower degradation of dietary CP. Therefore, the increased CP digestibility is much associated with the rumen contents of undegradable CP depending on the proportion of the DVR replacing SBM. According to Erasmus et al. (1994), degradability of CP is negatively correlated with amino acids outflow to small intestine. In addition, the DVR seems to be a source of ruminal undegradable CP fraction that coincides with low NH3-N formation and confirms the observed in vitro assay results.The roasted dry DVR may be effective in shifting degradation of CP from the rumen to the intestines without adverse effects on ruminal MCP synthesis which coincided with a decrease in NH3-N concentrations observed in vitro. Dry roasting vinasse rice could efficiently decrease ruminal solubility and degradability of CP and starch enhancing their escape to the stomach and the small intestine (Friedman, 1996) which could compensate the deficiency in amino acids profile in the diet. With respect to economic aspects, lambs fed on DVR diet shared much better BWG, ADG and FE resulting in 14 and 50% higher net profit and economic efficiency than control without compromising ruminal fermentation profile. Including DVR in the diet increased FE and consequently retained more energy resulting in higher BWG presumably due to decreased CH4 production as observed in vitro. The current results are desirable from the applicative and environmental points of view. Redirecting CH4 production towards more BWG is a principal interest of beef producers (Eckard et al., 2010) which form another advantage of DVR over SBM and may support its inclusion in sheep diets. From the applicative point of view, cost-effectiveness of DVR introduce it as a novel alternative source of protein suitable for ruminants diet formation and assures a new solution to animal nutrition challenges, particularly for developing countries. Increased MCP indicated that 50% replacement could alter the quality of CP entering the small intestine compared with the other replacement levels. The current in vitro results inferred that inlusion of DVR in the diet indicates more ruminal nutrients and fermentable energy supply adequate to enhance feed degradability with consequential increase in MCP synthesis. This emphasizes the improvement in lambs BWG and ADG which might be due to the potential complementary effect between DVR and SBM as described previously.
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5. Conclusions The partial replacement of soybean meal with 500 g DVR /kg had no adverse effects on feed intake and resulted in higher body weight gain, protein digestibility and net profit potentials. All these circumstances should certainly justify the novelty of using DVR which might be an alternative source of protein but also a source of energy in growing Texel lamb diets. However, future studies are needed to elucidate the effects of DVR on rumen microbes and their fermentability characteristics in commercial rations in long-term trials.
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